Recent advances in microbial and enzymatic engineering for the biodegradation of micro- and nanoplastics

This review examines the escalating issue of plastic pollution, specifically highlighting the detrimental effects on the environment and human health caused by microplastics and nanoplastics. The extensive use of synthetic polymers such as polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS) has raised significant environmental concerns because of their long-lasting and non-degradable characteristics. This review delves into the role of enzymatic and microbial strategies in breaking down these polymers, showcasing recent advancements in the field. The intricacies of enzymatic degradation are thoroughly examined, including the effectiveness of enzymes such as PETase and MHETase, as well as the contribution of microbial pathways in breaking down resilient polymers into more benign substances. The paper also discusses the impact of chemical composition on plastic degradation kinetics and emphasizes the need for an approach to managing the environmental impact of synthetic polymers. The review highlights the significance of comprehending the physical characteristics and long-term impacts of micro- and nanoplastics in different ecosystems. Furthermore, it points out the environmental and health consequences of these contaminants, such as their ability to cause cancer and interfere with the endocrine system. The paper emphasizes the need for advanced analytical methods and effective strategies for enzymatic degradation, as well as continued research and development in this area. This review highlights the crucial role of enzymatic and microbial strategies in addressing plastic pollution and proposes methods to create effective and environmentally friendly solutions.


Introduction
Plastic, rst introduced in the mid-20th century, has been essential to contemporary civilization due to its convenience and cost-effectiveness.Nevertheless, its extensive use has resulted in notable difficulties in waste management. 1 Projections indicate that the worldwide output of plastic might exceed 8.3 billion tons, possibly leading to a concerning 12 billion tons of garbage by the year 2050. 2 The buildup of waste in landlls and natural ecosystems poses signicant environmental issues.The prevailing pattern highlights the need for sustainable solutions, given that conventional approaches such as recycling and burning have shown their insufficiency, resulting in a substantial buildup of plastic trash in landlls. 3The magnitude of plastic manufacturing, coupled with inadequate waste disposal methods, has resulted in the pervasive presence of plastic trash and subsequent environmental pollution caused by microplastics, which are formed as a consequence of the degradation of larger plastic objects. 3,4These present substantial hazards to ecosystems, human well-being, and safety. 5,6The increasing recognition of this ecological problem emphasizes the pressing need for effective degradation remedies.
The widespread manufacturing and disposal of plastics have resulted in substantial contamination in land, water, and air environments, with microplastics being especially abundant.Inadequate waste management on land is a signicant cause of marine plastic pollution, leading to an estimated 5.25 trillion microplastics and nanoplastics entering seas, soil, and air. 7The presence of these minuscule particles is worrisome because of their detrimental impact on soil fertility and the well-being of marine organisms.Microplastics and nanoplastics have a profound negative impact on ecosystems and the health of species.They disrupt natural processes, damage animals, and accumulate toxic compounds in their organs. 8The presence of microplastics has the potential to affect the capacity of soil to allow water to pass through, its density, and the movement of nutrients, which raises worries about the consequences of their presence. 9,10The entrance of micro-and nanoplastics into oceans is inuenced by several processes, including UV photodegradation, mechanical forces, hydrolysis, and biological degradation. 11,12These minuscule particles disperse extensively across the ocean environment and have a notable inuence on marine life and ecosystems.Due to their diminutive size, they can effortlessly inltrate organisms and accumulate inside their organs, resulting in the accumulation of detrimental compounds that cause signicant hazards to marine life. 13he widespread use of articial polymers, which are crucial in contemporary society, has reached concerning levels and poses substantial ecological and physiological hazards in terrestrial, aquatic, and marine ecosystems.Due to their small size and large surface area, microplastics are very susceptible to absorbing harmful compounds including heavy metals, medicines, and ame retardants. 14Additionally, they are more easily consumed by living creatures.6][17][18][19] The heightened vulnerability of humans makes them particularly susceptible to the substantial risk posed by microplastic pollution.The pollutants have the ability to initiate inammatory and neurotoxic effects by activating certain protein kinase pathways.The effects of microplastics among various creatures differ depending on their ability to withstand environmental stress and the ecological circumstances in which they live. 20,21he extensive use of these polymers and their inclination to amass contaminants emphasize the need for devising efficient degrading techniques.The aim of these techniques is to disassemble microplastics and reduce their presence in the environment and alleviate the harm they cause (Scheme 1).
Plastics including polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC) have notable environmental difficulties since they degrade slowly in nature. 22he degradation pathways of these polymers may be classied according to their chemical composition: those with a carboncarbon backbone and those with heteroatoms in the main chain. 23,24The focus of this review is on the methodologies used by microorganisms to degrade synthetic polymers and the  specic roles of various enzymes in this biological process.These technologies provide effective methods for transforming plastic trash into carbon atoms, carbon dioxide, and valuable chemicals.The use of enzyme technology is crucial in promoting environmental conservation by converting plastic waste into less detrimental compounds. 25This review will explore the most recent developments in enzymatic degradation techniques and assess their potential in reducing the environmental and health consequences of plastic pollution, with a specic emphasis on microplastics and nanoplastics.

Enzymatic degradation challenges and progress in common synthetic polymers
The extensive use of synthetic polymers such as PET, PE, PS, PP, and PVC has played a substantial role in the global surge of plastic production, which reached a staggering 335 million tons in 2016. 26These materials, which are used in numerous industries, present signicant environmental obstacles because of their long-lasting and non-degradable characteristics. 4The increasing use of these polymers in our everyday lives calls for a careful evaluation of their environmental consequences, specically in terms of energy usage and waste production. 27his situation has prompted a greater focus on the development of sustainable alternatives and recycling methods.The pressing concern lies in nding innovative solutions that can mitigate the adverse effects of plastic proliferation, while maintaining a balance between the utility of these polymers and their environmental footprint.
The conversation surrounding the use of synthetic polymers revolves around their essential role in contemporary society and the resulting environmental hurdles.There are ongoing efforts to investigate environmentally friendly materials and improve recycling methods, but these endeavors encounter various obstacles, such as technological constraints and economic viability. 28The management of synthetic polymers' impact on the environment is a multifaceted challenge that necessitates a comprehensive approach.

Polyethylene (PE)
Dealing with polyethylene poses a serious challenge for enzymes when it comes to breaking down microplastics.This material is oen used in packaging and containers because of its outstanding ability to resist microbial degradation.PE, especially HDPE, possesses a linear structure that enhances its resistance to enzymatic degradation.Exciting advancements in biotechnology have revealed the incredible capabilities of specic enzymes and microbial strains in effectively binding to and breaking down PE. [29][30][31] Usually, these enzymes work by oxidizing the polymer chains, initiating a process of breaking them apart. 32Several environmental factors, such as exposure to UV light or physical abrasion, can contribute to this process. 33hese factors enhance the surface area to promote enzymatic activity.

Polyethylene terephthalate (PETE/PET)
Extensive research has been conducted on the enzymatic degradation of PET, which is commonly found in beverage bottles and textiles.8][39] The potential of enzymatic degradation of PET for recycling operations is immense and offers a more environmentally friendly and efficient option compared to traditional mechanical and chemical recycling methods (Fig. 1).

Polypropylene (PP)
PP is widely used in packaging and automotive components due to its impressive resistance to a range of chemical degradation processes. 40,41However, a recent study has uncovered the potential of enzymatic techniques in degrading PP.Certain microbial strains have been acknowledged for their remarkable capacity to adhere to and partially degrade PP. 42,43 The challenge is to enhance the efficiency of these biological processes to make them more practical for widespread use.A study is being conducted to explore and improve enzymes that have the potential to enhance the efficiency of breaking down the chemical bonds in PP. 44 Polyvinyl chloride (PVC) PVC, widely used in the construction and packaging industries, poses challenges for biodegradation because of its chlorine content. 45There is a lack of comprehensive scientic knowledge regarding the enzymatic degradation of PVC, especially when  compared to other polymers.There has been a strong focus on identifying and managing microbial strains that can tolerate and break down the chlorine-based compounds commonly found in PVC. 46,47The study in this area is still in its initial phases with the goal of developing dependable and effective methods for the secure breakdown of PVC.

Polystyrene (PS)
PS, a widely used material in packaging and insulation, is wellknown for its remarkable durability due to its chemically stable aromatic structure. 48,491][52] The enzymes effectively break down the styrene monomers, and the current pace and efficiency of the process meets expectations. 53The present study aims to enhance the efficiency and longevity of these enzymes to make the biodegradation of PS a more practical option.

Polylactic acid (PLA)
PLA, a bioplastic derived from renewable sources, is inherently more prone to enzymatic degradation when compared to polymers made from petroleum. 546][57] The degradation of PLA happens through the hydrolysis of ester bonds, leading to the formation of lactic acid, which can be subsequently metabolized by various microorganisms. 58PLA stands out for its positive environmental impact since it can be easily and completely broken down in composting facilities.

Polybutylene succinate (PBS)
It has been observed that PBS, a type of biodegradable plastic, is more susceptible to enzymatic degradation compared to conventional polymers. 2,59There have been signicant ndings in the eld of enzyme research, particularly in the area of PBS degradation. 60,61Notably, lipases have demonstrated remarkable efficiency in breaking down PBS.The degradation process involves the hydrolysis of ester bonds in the polymer, causing it to break down into smaller particles that can be easily broken down by biological processes. 62Because of its remarkable vulnerability to degradation, PBS is an extremely appealing material for endeavors that prioritize ecological sustainability.

Polyurethane (PUR)
Enzymatic degradation of PUR can be quite complex due to its various chemical compositions. 63,64However, certain enzymes such as esterase and ureases show great potential in breaking down PUR. 28,65,66 The enzymes have the ability to specically target certain bonds in the PUR polymer, resulting in its degradation. 28Researchers in this eld are focused on uncovering and enhancing the efficiency of these enzymes with the goal of developing effective techniques for breaking down PURs.The signicance of this issue stems from the widespread use and persistent presence of PURs in the environment.

Inuence of chemical composition on plastic degradation dynamics
8][69] These factors encompass the intrinsic characteristics of the plastic, such as its molecular structure, composition, physical form, and the inclusion of additives. 23,70,71The rate and mechanism of biodegradation are greatly inuenced by these characteristics.Note that the susceptibility of a plastic to enzymatic attack can be signicantly affected by the presence of specic functional groups or the degree of polymer branching.
Factors such as pH, temperature, oxygen levels, and light exposure are critical in inuencing the effectiveness of enzymatic degradation.3][74][75] Environmental factors can also weaken the structure of plastics, making them more susceptible to enzymatic breakdown.
The fundamental chemical composition of plastics, specically the types of bonds they possess, plays a crucial role in determining their degradability.Plastics with carbon-carbon (C-C) backbones, such as polyethylene and polypropylene, have a remarkable ability to resist microbial and enzymatic decomposition, which signicantly slows down the degradation process. 76On the other hand, polymers that incorporate heteroatoms into their main chain, such as polyesters, are highly prone to enzymatic hydrolysis. 77he interaction between degrading enzymes and a plastic is greatly inuenced by the chemical composition, which determines the surface hydrophobicity.Surfaces that repel water can also cause hydrophilic enzymes to be repelled, which presents initial obstacles in the biodegradation process.Effective enzymatic binding and action oen require the formation of a biolm. 78,79iven the origin of most modern plastics from petrochemical sources, the widespread existence of non-biodegradable plastics presents a notable environmental concern. 13To tackle this problem, it is important to keep working on improving our understanding and nding ways to enhance the degradation of microplastics by enzymes.Adapting enzymatic methods to effectively tackle the distinct obstacles posed by various plastic polymers is needed for advancing sustainable waste management and environmental conservation strategies.

Examining the enduring environmental effects of microplastics and nanoplastics
It is essential to have a comprehensive understanding of the physical characteristics and long-term effects of microplastics (MPs) and nanoplastics (NPs) in order to effectively tackle the ongoing issue of these pollutants in various ecosystems.The extensive variety of synthetic polymers used in the composition of MPs results in a wide range of physical forms, including foam, pellets, akes, bers, and lms.1][82][83][84] Nanoplastics possess distinct characteristics in comparison to MPs as a result of their reduced dimensions.0][91] The widespread pollution poses considerable threats to multiple aspects of the ecosystem, such as the food chain, plant life, marine organisms, and human populations. 92,93This pollution greatly affects both humans and the environment, as they both experience the consequences of it.The structural composition of polymers plays a key role in determining their environmental behavior and has a combination of ordered and disordered regions that have a signicant impact. 23The current situation underscores the need for effective methods to address these persistent pollutants.

Exploring the ecological and health implications of microplastic and nanoplastic pollution
5][96] These particles have been associated with serious health risks, such as the potential to cause cancer and interfere with endocrine systems. 97,98Their environmental impact is exacerbated by their capacity to attract and transport other detrimental substances, including persistent organic pollutants and heavy metals. 99,100In addition, MPs and NPs can carry harmful bacteria, including antibiotic-resistant strains, which poses a considerable threat to both wildlife and human communities.
The management and regulation of MPs and NPs require advanced analytical and quantitative methods, particularly when addressing complex environmental matrices.The challenge is made more difficult by the complexity of recycling these particles. 101A deep understanding of the physical and chemical properties of MPs and NPs is needed to develop effective strategies for their enzymatic degradation.
In order to effectively tackle the negative impacts of MPs and NPs, it is important to acknowledge their wide-ranging implications and establish dependable approaches for their identi-cation, assessment, and breakdown.This eld of study not only provides potential solutions for addressing the environmental and health risks linked to microplastic and nanoplastic pollution, but also emphasizes the importance of ongoing efforts to comprehend and combat this widespread problem.

The role of microbes in plastic degradation
3][104][105][106] This area of study investigates the capacity of different microorganisms and insects to transform plastic into eco-friendly substances. 92,93][109] These microorganisms, found in various habitats such as soil, aquatic environments, and even air, have an important role in the natural breakdown of organic matter.An excellent illustration is the breakdown of PE, a commonly used plastic, by a range of microorganisms. 29,31One of the most fascinating ndings in this eld involves the gut bacteria found in the larvae of the large waxworm, Galleria mellonella. 110,111These bacteria have demonstrated impressive effectiveness in breaking down PE through a process known as hydrolysis. 112his research on the interaction between microbes and plastic has uncovered exciting possibilities for addressing plastic waste.It has the potential to transform discarded petroleum-based polymers into reusable materials or feedstock for biomass production, and offers new ways to manage plastic waste effectively. 24,113The success of these groundbreaking solutions depends on a comprehensive grasp of the distinct microbial enzymes implicated in the degradation process.A deep understanding of this subject is crucial to create more efficient and sustainable approaches to tackle the growing issue of plastic pollution and advance the eld of enzyme-based microplastic degradation.

Dynamics of the microbial degradation of plastics
The breakdown of microplastics (MPs) by microorganisms is a sophisticated process driven by enzymes.Many factors can inuence the process, including the molecular weight and chemical composition of the microplastic, environmental conditions, the specic microbial species, and the physical properties of the plastic, such as crystallinity and the presence of functional groups or additives. 8,9The degradation process usually occurs in a sequence of stages beginning with the development of a biolm on the surface of the plastic.This is then followed by biodegradation, biotransformation, and nally mineralization. 35,114Irrespective of the natural degradability of the polymer, the rst step involves hydrolysis, which breaks down microplastics into smaller molecular fragments.This step is crucial and relies on the microorganisms at hand (Fig. 2).
Microorganisms play a key role in the intricate biochemical reactions that occur during the aerobic biodegradation of plastics.Microorganisms use oxidizing enzymes to generate carbonyl groups, which are subsequently oxidized into carboxylic acids. 115This results in the hydrolysis of the polymer chain, which facilitates degradation.The microorganisms metabolize the small hydrocarbon fragments produced in a highly efficient manner. 116The last step involves converting the hydrolysis products into microbial biomass, resulting in the release of water and carbon dioxide. 93,117The enzymes involved in this degradation process can be classied into two main categories: those that alter the surface of microplastics to enhance their solubility in water, and those that break down the plastic into smaller components for microbial metabolism. 118n understanding of the intricate processes involved in the breakdown of microplastics by microorganisms is needed to devise successful approaches to minimize the negative effects of microplastic pollution on the environment.It is also crucial for making progress in enzyme-based techniques for microplastic degradation and gaining valuable insights into potential solutions for this urgent environmental issue (Table 1).

Determinants of microbial efficacy in plastic degradation
The degradation process of microplastics is inuenced by a variety of factors including microbial growth kinetics, the properties of microplastics themselves, and the environmental conditions at hand.The structural properties of microplastics, their material composition, shape, and the presence of additives play a main role in determining their vulnerability to biodegradation.
External environmental factors, including pH levels, temperature, oxygen availability, exposure to light, and the presence of other substances, are crucial in inuencing the process.The interaction between temperature and pH is key since pH levels have a signicant impact on the electrostatic interactions between the microplastic surface and microorganisms and chemicals in soil or water. 130Degradation can be slowed down under certain conditions, while enzyme activity may be affected in different environments.For the most effective biological degradation of microplastics, it is important to maintain optimal conditions such as low pH levels and low temperatures.The rate of degradation can be inuenced by a range of chemical and physical properties of microplastics, including density, molecular weight, degree of crystallinity, and the presence of specic functional groups or substituents. 131icroplastics with carbon-carbon (C-C) bonds provide enhanced durability against microbial attack, whereas microplastics with ester bonds are more prone to the effects of hydrolytic enzymes. 116he inclusion of plasticizers or other additives can greatly affect the biodegradability of microplastics.These additives have the potential to either enhance or impede microbial colonization depending on their unique characteristics and the composition of the microbial community they are targeting. 116The presence of external substances on the surface of microplastics can slow microbial degradation.On the other hand, nutritional supplements that are high in carbon and nitrogen can enhance the growth of microorganisms on microplastic surfaces, thereby speeding up the degradation process. 131These different factors are important in the development of enzyme-based approaches for efficient microplastic degradation (Fig. 3).

Evaluating the effectiveness and challenges of biological plastic degradation
Progress in the degradation of plastics by microorganisms, particularly with enzyme-based methods, has been impressive.Biocatalysts have demonstrated signicant potential in breaking down microplastics (MP) into smaller particles such as nanoplastics (NP).There is increasing interest in these biocatalysts due to their ability to greatly decrease the size of plastic particles.Nevertheless, the efficiency of microorganisms in degrading plastics is inuenced by the various characteristics of the materials and is contingent upon the size of the plastic particles, ranging from larger akes to minuscule dimensions. 134everal biodegradation methods are currently under investigation, such as pure bacterial cultures, fungal cultures, bacterial consortia, and the use of specic enzymes. 135Microorganisms such as bacteria, fungi, and algae have shown the ability to break down complex plastic polymer chains into smaller units, or monomers. 136,137This ability is derived from the production of certain enzymes or metabolites that aid in the degradation process.One of the difficulties of microbial degradation is the considerable amount of time needed to break down contaminants.Even with the demonstrated efficacy of bacteria, the process of breaking down microplastics can be time-consuming in certain instances and oen takes several months.Efforts are currently being made to enhance environmental conditions and optimize microbial strains to accelerate the degradation process and improve the efficiency of bacterial action on MPs and NPs.In light of the difficulties posed by degradation rates, there are continuous endeavors to investigate the thorough biodegradation of microplastics and nanoplastics.

The crucial role of microorganisms in plastic degradation
The issue of plastic pollution has become more pressing, and the development of creative and environmentally friendly solutions, particularly in the area of plastic degradation, is needed.Enzymatic methods for plastic degradation have become increasingly recognized as very effective and ecofriendly alternatives to traditional plastic treatment methods.This strategy, grounded in a commitment to environmental sustainability, offers a way to transform plastics into reusable monomers or create valuable bioproducts by converting them into carbon dioxide, water, and new biomass. 138,139icroorganisms are crucial in this process since they produce specialized enzymes that can effectively break down polymers and support the metabolism of the resulting hydrolyzates.The enzymatic biodegradation of polymers usually involves important reactions such as hydrolysis and oxidation, which break down polymer chains into smaller oligomers and monomers. 140,141These reactions are essential for the breakdown of various polymer bonds, such as ester, carbonate, amide, and glycoside bonds, resulting in the creation of monomers.In terms of their susceptibility to enzymatic degradation, petroleum-based polymers can be categorized into two types: hydrolyzable and non-hydrolyzable.Examples of hydrolyzable polymers include polyethylene terephthalate (PET) and polyurethane (PUR), while non-hydrolyzable polymers include polyethylene (PE), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC). 141,142Enzymes from the hydrolytic enzyme group, including esterases, lipases, depolymerizers, and fetases, have a high prociency in breaking down the carbon structure of plastics.They focus on the ester bonds and carbonyl carbon atoms that are created when the oxidation process occurs, transforming the polymer into individual monomers. 143evertheless, the degradation of non-hydrolyzable polymers such as PE, PS, PP, and PVC poses a considerable obstacle.The chemical degradation of the carbon backbone is a complex area that still requires further research.Ongoing progress in enzymatic degradation is needed to preserve the environment and improve recycling techniques.

Enzyme specicity in plastic polymer degradation
In the area of enzyme-mediated microplastic degradation, past research has revealed the involvement of numerous microorganisms and enzymes in the breakdown of polyethylene (PE).][147] The degradation mechanisms for various forms of PE, including LDPE and HDPE, are currently being studied.Enzymes like laccase and alkane hydrolases have demonstrated potential in initiating PE degradation.Laccase, in particular, aids in oxidizing the PE surface and creating carbonyl regions that are more prone to subsequent enzymatic activity.Alkane hydrolases have shown great efficacy in breaking down heat-treated PE. 135,[148][149][150][151] Enzymes involved in PE degradation have been isolated from a wide range of microbial sources, including Proteobacteria, Firmicutes, and Actinobacteria.These microorganisms have shown promise in aiding the breakdown of microplastics.However, there is still much to learn about the exact process by which these enzymes degrade microplastics, and further research is needed. 152urthermore, there have been notable advancements in the eld of microbial enzymes that possess the ability to break down lignin polymers containing oxidative C-C bonds.Notable examples include manganese peroxidase, lignin peroxidase, and laccase.The understanding of these enzymes in the area of polyethylene biodegradation is continuously developing, and additional research is necessary to gain a complete grasp of their functions and mechanisms.It is important that future studies focus on gaining a deeper understanding and characterization of the different enzymes involved in polyethylene degradation. 153,154cused enzymatic activity targeting polyethylene terephthalate The eld of enzymatic microplastic degradation has made signicant progress, especially in targeting polyethylene terephthalate (PET).This is based on extensive research on Ther-mobida fusca hydrolase, which has led to the discovery of various enzymes that can break down PET.Out of all the enzymes, the PETase enzyme from Ideonella sakaiensis 201-F6 is particularly 142 noteworthy for its impressive capacity to effectively degrade PET into various intermediates such as BHET, MHET, and TPA.MHETase, an additional enzyme, carries out further processing of MHET to produce terephthalic acid and ethylene glycol. 34,155ecent advancements have resulted in the creation of stronger versions of PETase, which have improved stability and efficiency in breaking down materials.In addition, a 25 kDa suberinase from Streptomyces scabies has demonstrated considerable potential in the degradation of PET. 156,157Thorough analyses of the structure and mechanisms have uncovered distinct interactions between PETase, cutinase, and PET, which occur through an induced t mechanism.PETase is capable of breaking down a wide range of polycyclic aromatic microplastics due to its impressive versatility. 158,159fforts to enhance the efficiency of PETase have involved various techniques such as mutagenesis, overexpression, and the use of microalgae for transformation.These advancements are crucial in tackling the environmental issues caused by PET pollution.This research is a signicant advancement in developing enzymatic methods to effectively break down PET microplastics and makes a valuable contribution to environmental remediation endeavors. 160,161Enzymatic strategies for polystyrene degradation Important advancements have been made in the eld of enzymatic microplastic degradation, with a particular focus on polystyrene (PS).Progress in this eld is evident from the fact that a wide range of microorganisms, such as bacteria and fungi, have the ability to break down PS.Nevertheless, the precise enzymes responsible for initiating this degradation process remain to be fully understood. 135,162rior research has emphasized the signicance of extracellular esterases from Lentinus tigrinus in the breakdown of PS.6][167][168][169] These enzymes are essential for the conversion of PS polymers through a series of reactions.It starts with the transformation of monomers into styrene, followed by oxidation to phenylacetate, and ultimately the integration of phenylacetate into the Krebs cycle. 170n in-depth knowledge and thorough analysis of these enzymes is essential to enhance the enzymatic breakdown of PS microplastics.This research explores new possibilities for tackling the environmental issues caused by PS waste and offers creative and eco-friendly solutions for managing PS waste.Efforts to identify and optimize these enzymes are needed to develop effective strategies to address the impacts of PS pollution.

Advancing enzyme engineering for plastic degradation
Strategies for enhancing stability and efficacy of enzymes in plastic degradation.Advances in enzyme engineering are necessary to effectively break down microplastics.An important aspect in this eld is to enhance the stability and activity of enzymes, especially those involved in breaking down plastic materials. 171his entails leveraging structural similarities between various enzymes to enhance their functional properties, typically with the application of site-directed mutagenesis techniques. 172nother important development involves enhancing the heat tolerance of enzymes that target plastics.This is particularly important for plastics with high glass transition temperatures (T g ), as their crystallinity decreases as the temperature rises. 173his can make it easier for enzymes to access the plastics and speed up degradation.Plastics are easier to process by enzymes when they become more exible and mobile at temperatures near or above their T g . 173Nevertheless, a serious challenge arises when it comes to naturally occurring enzymes such as PETase since they tend to lose their efficiency when exposed to high temperatures, which restricts their ability to maintain thermal stability. 174To tackle this problem, previous studies have primarily concentrated on developing different versions of PETase and similar enzymes that possess the ability to endure elevated temperatures, thus aligning with the T g of various types of plastics.This requires a thorough examination of the distinctive characteristics of thermophilic proteins and using this knowledge to enhance the ability of plastic-degrading enzymes to withstand high temperatures.The ultimate objective is to maximize the efficiency of these enzymes, particularly in environments that closely align with the T g of the specic plastic being targeted. 13,122ith the development of enhanced enzymes, we can create enzymes with greater power and efficiency that enables them to break down a wider variety of plastics.Enhancement of the thermal stability and pH tolerance of these enzymes has the potential to signicantly improve their practicality in environmental cleanup and recycling processes.Continual endeavors in enzyme engineering focus on surpassing the constraints of natural enzymes, while also customizing enzymes to full precise needs for plastic degradation. 175Our work involves enhancing enzymes to target specic types of plastics, enhancing their selectivity, and maximizing their catalytic efficiency.The improvement of the stability and activity of enzymes is crucial for the progress of enzyme-based microplastic degradation.

Enhancing enzymatic thermal stability through the use of disulde bonds for microplastic degradation
The enhancement of enzyme thermal stability has become a key method in enzymatic microplastic degradation, particularly in the area of PET hydrolysis.One effective approach involves incorporating disulde bonds and salt bridges.This process requires meticulous adjustment of the protein structure at specic locations or in its overall arrangement.As an illustration, the replacement of amino acid residues in metal-binding regions with disulde bonds has been demonstrated to greatly improve the ability of enzymes to withstand high temperatures. 176he signicance of disulde bridges is especially notable in PET hydrolases, which possess numerous sites for binding divalent metals, as evidenced by the crystal structure of the Cut190 enzyme.It has been noted that the inclusion of divalent ions such as calcium (Ca 2+ ) or magnesium (Mg 2+ ) has the dual effect of enhancing the thermal stability of the enzyme and optimizing its temperature range for operation.Methods such as circular dichroism (CD) have shown that the melting temperature of the enzyme is signicantly raised with the addition of calcium ions.Further insights from molecular dynamics (MD) simulations and X-ray structural analysis have uncovered the signicance of Ca 2+ binding in triggering essential conformational alterations in the enzyme, which results in enhanced catalytic efficiency. 122The presence of intramolecular disulde bridges, specically DS1 and DS2, plays an important role in maintaining the functional integrity of the catalytic triple bond in enzymes such as PETase.One aspect that enhances the exibility of the loop is DS1, which leads to a boost in enzyme activity.On the other hand, DS2 is crucial in preserving structural stability.Focused enhancement of these sites has been demonstrated to greatly improve the capacity of the enzyme to break down PET. 177,178 Investigation of the crystal structures of different enzymes, such as LCC, Tf cutinase, and IsPETase, has opened up possibilities for developing innovative approaches to enhance their resistance to heat.As an illustration, enzyme activity has been enhanced by replacing the divalent metal binding site of LCC with a disulde bridge and introducing targeted mutations. 179Currently, there are ongoing efforts to enhance the thermal stability of enzymes that play a role in breaking down plastics, specically PET.This task requires the generation of enzyme mutants using directed evolution techniques, as well as the identication of optimal sites for calcium binding and disul-de bridge formation.
The strategic use of disulde bonds is demonstrating the enhancement of the thermal stability of enzymes employed in the degradation of microplastics.These advancements are crucial for the development of stronger and more effective enzymatic solutions to tackle the environmental issues caused by plastic pollution.

Advancements in enzymatic stability through hydrogen bonding and electrostatic interactions
Signicant progress has been achieved in enhancing the stability of protein structures with a dedicated emphasis on hydrogen bonding and hydrophobic interactions.The advancements in the strategic use of proline and its surrounding residues are very noteworthy.Prolines, known for their unique cyclic side chains, have played an important role in enhancing the structural rigidity and thermal stability of PET hydrolases.
The substitution of serine with proline in bacterial PET hydrolases such as Est119 from Thermobida alba AHK119 and Cut190 from Saccharomonospora viridis AHK190 results in a notable enhancement in heat resistance and PET degradation efficiency.In the same way, the incorporation of proline into enzymes such as LCC ICCG tetramer and Thermobida alba cutinase has a notable effect on their melting temperature and boosts their hydrolytic activity toward PET.Enhancement of the hydrogen bonding network in enzymes has been a key area of study, and PETase is a prominent illustration of this.Modications made to the exible regions of these enzymes have led to the development of variants that exhibit enhanced rigidity and thermal stability. 176,180Notable examples include the IsPETase S121E/ D186H double variant and the ThermoPETase triple variant.These variants exhibit higher melting temperatures and stronger binding to PET, which enhances their suitability for industrial applications. 181FAST-PETase mutants, such as S132E, D186H, R224Q, N233K, and R280A, have been found to exhibit exceptional kinetics and performance at elevated temperatures, surpassing the capabilities of the original enzyme in terms of hydrolysis efficiency.Crystal structure analysis of these mutants has conrmed their noteworthy contribution to enhancing the thermal stability of the enzyme. 182evertheless, challenges persist in breaking down highly crystalline plastics, which hinders the extensive use of these enzymes.Research has indicated that FAST-PETase can fully break down PET aer thermal pretreatment, suggesting that these enzymes are successful in the recycling process. 182owever, their use in the environmental degradation of crystalline polymers is still somewhat restricted.

Enhancing enzymatic stability with glycosylation in microplastic degradation
Enhancement of the thermal stability of enzymes used in microplastic degradation has gained signicant attention, and glycosylation has emerged as a promising technique in this regard.This process has demonstrated encouraging results in enhancing thermal stability, particularly when used with enzymes expressed in eukaryotic microbial cells.Understanding glycosylation, a crucial post-translational modication, is essential to maintain protein stability and prevent thermal aggregation.
An excellent illustration is the PET hydrolase LCC, renowned for its exceptional thermal stability.The expression of LCC in Pichia pastoris led to glycosylation, which resulted in an enzyme form that exhibits increased resistance to high-temperature aggregation.This variant of LCC has enhanced efficiency in breaking down PET at elevated temperatures. 183Nevertheless, achieving precise control over the glycosylation sites on the enzyme's surface poses a serious challenge, given its potential impact on the interaction of the enzyme with the PET substrate.Precise positioning of these glycosylation sites is needed to prevent negative impacts on the active site and overall functionality of the enzyme.Cutting-edge computational methods, such as GRAPE, have been employed to enhance enzymes such as IsPETase, leading to the development of variants like Dura-PETase.These variants exhibit a remarkable increase in melting temperature, thereby enhancing their stability and effectiveness in degrading PET. 184n a previously reported study, neural networks were used to conduct extensive in silico mutagenesis and experimental validation.The aim was to develop enzymes with enhanced thermal stability.During this study, a number of mutations were discovered in PETase that greatly enhanced its performance in high temperature conditions.The enzyme FAST-PETase demonstrated a notable enhancement in the rate of hydrolysis when compared to its original form.While it is important to consider the potential impact on the catalytic efficiency of the enzyme, 182 it is also benecial to increase thermal stability.The function of the enzyme can be inuenced by structural modi-cations in the active site.As an illustration, replacing Ala with Arg280 in PETase enhances the speed of PET degradation.
Glycosylation is a crucial element in enhancing enzyme thermal stability, and it is essential to carefully select a glycosylation site that does not disrupt the catalytic activity of the enzyme.A meticulous approach to enzyme modication is needed for the advancement of enzyme-based strategies for microplastic degradation (Fig. 5).

Enhancing microplastic degradation through improved enzyme-substrate dynamics
The effectiveness of enzyme-based microplastic degradation relies heavily on the intricate interactions between enzymes and their specic substrates, particularly when it comes to PET hydrolases.This aspect is of the utmost importance in heterogeneous catalysis because it involves the solubility of the enzyme in an aqueous system, which is in stark contrast to the insolubility of the PET chains. 187,188This imbalance frequently results in substantial adsorption on the PET surface, which can have a negative impact on the catalytic efficiency of the enzyme. 189It is worth noting that the structure of PETase does not inherently facilitate substrate binding.This discovery has sparked a surge of scientic curiosity in hydrophobic polymers that resemble naturally occurring substances such as carbohydrate-active enzymes.The interaction between enzymes and PET substrates is mainly inuenced by the electrostatic and hydrophobic interactions among the amino acid residues. 190,191he improvement of the interaction between enzymes and substrates is a main area of emphasis.One efficient approach is to alter the hydrophobic surface and/or electrostatic properties of the enzyme.Through an analysis of the charged and solventexposed amino acid residues of proteins, studies are striving to minimize the electrostatic repulsion between the PET substrate and the enzyme surface. 121,192,193These adjustments can enhance the binding affinity and boost the degradation efficiency of PET.These advancements are crucial in the development of enzyme-based methods for microplastic degradation and have a signicant role in reducing the environmental impact of plastic pollution.
The enhancement of enzyme-substrate interactions is not just a scientic endeavor, but an important aspect of promoting environmental sustainability.Through careful optimization of these interactions, enzymes can be customized to better target and break down particular forms of microplastics, resulting in the higher overall efficiency of the degradation process.This approach shows immense potential in addressing the growing concern of microplastic pollution and offers a more efficient and eco-conscious solution to this worldwide problem.With the ongoing advancements in research, the potential for developing highly effective and specialized enzymes for microplastic degradation is growing.

Enhancing the active site of PET hydrolase to enhance microplastic degradation
The enzymatic degradation of polyethylene terephthalate (PET) microplastics is intricately linked to the interaction between the active site of PET hydrolases and the PET substrate. 194In order to optimize the catalytic activity of these hydrolases, it is oen necessary to make precise modications in the active site region.These modications may involve reconguring the substrate, adjusting the cofactor specicity, or introducing mutations in the active site that have been proven to greatly affect the overall reactivity. 195,196One important objective in PET-degrading enzyme engineering is to enhance the accessibility of the active site to the plastic surface. 197This is usually achieved by broadening the range of substrates that can bind to the enzyme.The approach used involved manipulating Fusarium solani cutinase to create the L182A mutant.This mutant was designed to have enhanced hydrolytic activity against PET by making specic amino acid modications to expand the active site niche. 198nzymes such as PETase, Cut190, MHETase, and Pseudomonas aestusnigri hydrolase have employed comparable techniques to selectively target residues in the substrate binding site by means of structural mutagenesis. 199Recent modications have signicantly enhanced the binding of PET substrate and minimized the interference caused by degradation byproducts.As a result, the efficiency of PET depolymerization has been greatly improved. 200Efforts are currently being made to enhance the hydrophobic characteristics of the binding site or optimize the active site.][203] In addition, modifying the structure of the active site of the enzyme can assist in reducing the inhibition caused by PET degradation intermediates or products. 204These different strategies work together to improve the interaction between PET hydrolase and its substrate for more efficient degradation of PET.This is a signicant contribution to tackling the environmental issues associated with microplastics made from PET.The improvement of the active site of PET hydrolases is a crucial step in the development of more efficient enzymatic solutions for microplastic degradation (Table 2).

Enhancing enzyme surface properties to enhance efficiency in degrading microplastics
In the area of enzyme-based microplastic degradation, numerous endeavors have been undertaken to enhance the efficiency of plastic degradation.This involves modifying the surface properties and charge characteristics of the active site of   PET hydrolases.The enhancement of the hydrophobicity of the active site has been shown to be a successful approach to improving binding to plastic substrates and consequently boosting degradation efficiency.As an illustration, when the cutinase Cbotu_EstA is adjusted, 214 it reveals a greater portion of its hydrophobic surface.This leads to improved adsorption to PET substrates and an increase in hydrolytic activity.Nevertheless, an overabundance of hydrophobic residues may result in undesirable consequences like enzyme aggregation or structural instability.
In a previously reported study, certain mutations that greatly improve the activity of PETase were discovered.This was achieved with the use of molecular docking and crystallographic analysis.Several mutations, including R61A, L88F, and I179F, have been found to greatly enhance enzyme efficiency.In a similar vein, modication of the thermostable LCC and Tf Cut2 PET hydrolases by substituting His/Phe with Ser/Ile enhances their ability to break down PET at lower temperatures, resulting in more efficient depolymerization. 202The improvement of hydrophobicity can also enhance enzymesubstrate interactions.This was observed in the PHB-degrading enzyme from R. pickettii T1, where the substitution of serine and tyrosine with hydrophobic residues increased adhesion to the PHB surface.As a result, the efficiency of plastic hydrolysis was signicantly improved. 223ecent developments in enzyme design have led to notable improvements in catalytic activity and a reduction in byproduct inhibition.For instance, the Tfu_0883 cutinase underwent a double mutation (Q132A/T101A), resulting in notable enhancements.The replacement of amino acid residues in the active site of the TfCut2 cutinase with residues from the LCC cutinase led to enhanced PET degradation at higher temperatures. 203Furthermore, the 192 substitution of a mutation, Ile179, in the PETase enzyme with the more hydrophobic Phe resulted in an enhanced catalytic efficiency toward PET substrates at 30 °C. 202The modications made enhanced the alignment of the binding sites and resulted in more robust interactions between the enzyme and substrate.These recent advancements underscore the signicance of thoughtfully planned enzyme modications in enhancing the breakdown of PET and other plastic materials.These strategic modications in enzyme surface properties demonstrate promise for developing more efficient solutions to the urgent issue of microplastic pollution. 185,192,213proving enzyme functionality with the use of accessory binding domains In order to enhance the efficiency of enzyme-based microplastic degradation, especially for PET, previous studies have investigated the integration of accessory binding domains that draw inspiration from the intricate structure of cellulases.These auxiliary modules, referred to as carbohydrate-binding modules (CBMs), are segments present in carbohydrate-active enzymes that facilitate the breakdown of natural biopolymers.The integration of CBMs into PET hydrolysing enzymes seeks to optimize the interaction of the enzyme with PET, leading to improved degradation efficiency.CBMs are highly regarded for their excellent compatibility with a wide range of natural polymers and synthetic plastics.Nevertheless, predicting the protein sequences that determine the function of PET-binding modules poses a serious challenge due to their inherent complexity. 224,225otable advancements have been achieved in this eld, such as the successful combination of a cutinase from Thermobida fusca with CBMCenA from Cellulomonas mi to enhance the breakdown of PET bers. 216An alteration was made to CBMCenA by introducing a single tryptophan mutation.This modication aimed to enhance its adherence to PET bers and enable more efficient enzymatic degradation. 226Moreover, the combination of Thc_Cut1 cutinase from Thermobida cellulosiliqua and CBM trCBH from Hypocrea jecorina notably boosts the binding of the enzyme to the PET surface, thereby enhancing its capacity to break down the material.The combination of PET hydrolases and CBM has demonstrated encouraging outcomes in enhancing the efficiency of PET degradation in various PET feedstocks. 227dditional cutting-edge techniques being investigated involve the use of polyhydroxyalkanoate binding modules (PBMs), hydroponics, and amphiphilic anchor peptides for chimeric fusion.These methods focus on enhancing the attachment of enzymes to PET to improve the degradation of polyester-PU nanoparticles.As an illustration, the combination of hydrophobic, a protein with hydrophobic properties, and PETase has proven to be effective in improving the binding and degradation of PET lms.These strategies, which aim to improve the binding ability of PET-degrading enzymes, show signicant potential in optimizing PET binding and enhancing hydrolysis efficiency.This offers a fresh approach to tackling the issue of microplastic pollution. 72,215,217,219,220ctors inuencing the efficiency of enzymatic plastic degradation In order to gain insights into the enzymatic degradation efficiency of microplastics, an extensive analysis was carried out to examine the distinctive properties of plastic polymers.The chemical structure, molecular weight, and crystallinity of these polymers play a crucial role in determining their degradation rate.Polymers containing ester bonds, such as polyester polyurethane, typically demonstrate greater biodegradability in comparison to polymers lacking these bonds. 228Biodegradation of high molecular weight plastics can be more challenging, but there are additives available that can assist in the process.The intricate composition of microplastics, with their symmetrical shapes, strong hydrogen bonds, and regular units, can oen impede enzymatic degradation. 229nvironmental factors are also inuential in the degradation process.Temperature is important since elevated temperatures can speed up degradation and affect oxidation mechanisms.Accurate pH levels are also important in assessing the activity and growth of microorganisms that contribute to degradation.Additionally, different pH levels can affect the structure of plastic and its vulnerability to degradation.UV exposure and biodegradation are key factors contributing to the breakdown of plastic.Other factors, such as mechanical shredding, temperature, pH, and catalysts, also play a role in this process.Humidity plays a signicant role in the biodegradation process.][232] The degradation kinetics of plastics are inuenced by a variety of factors, resulting in a complex and multifaceted degradation process.This review highlights the signicance of creating efficient enzyme-based strategies that are customized to the distinct characteristics of various microplastics to enhance their degradation efficiency.

Tackling PET pollution with enzymatic solutions
Degradation process of PET by hydrolytic enzymes Polyethylene terephthalate (PET), a widely used synthetic plastic found in disposable beverage containers, has experienced a notable surge in global production.In 2013, a staggering 56 million tons of PET were manufactured. 233The durability of PET, stemming from its aromatic ring structure and ester bonds, plays signicant role the issue of plastic pollution.Improper disposal of single-use plastics further worsens this problem.In contrast to other biodegradable polyesters such as polyhydroxyalkanoate, PCL, polybutylene succinate, and poly(butylene adipate-coterephthalate) (PBAT), PET is recognized for its resistance to natural degradation processes.5][236] One such microorganism, Ichneumonella sakaiensis 201-F6, has demonstrated the ability to utilize the terephthalate component in its metabolic activity.This discovery offers a fresh perspective on the degradation of PET (Fig. 6). 34 study involving Pseudomonas putida GO16 highlights the signicant progress made in biotechnology, specically in converting PET into more environmentally friendly materials such as polyhydroxyalkanoates.This process, which uses pyrolysis, demonstrates potential for the recycling of PET waste. 237The degradation rate of PET lms is inuenced by various factors including crystallinity, purity, and the orientation of the polymer chains.These factors greatly affect the efficiency of the degradation process.PET microplastics present a serious environmental concern due to their potential effects on human health, specically in relation to the endocrine system and estrogen regulation. 238,239This situation highlights the urgent demand for innovative and efficient degradation and recycling methods for PET and other synthetic polymers.Enzyme-based strategies for microplastic degradation have gained recognition as a viable solution, providing a sustainable approach to address the environmental consequences of PET pollution.

Enhancing enzyme technology for efficient PET degradation
Remarkable advancements have been achieved in enhancing the performance of PETase, the key enzyme in the breakdown of polyethylene terephthalate (PET), in the area of enzyme-based microplastic degradation.Prior research has primarily concentrated on enhancing the interaction of PETase with PET substrates. 240,241As an illustration, PETase was modied with double mutations to enhance the efficiency of PET degradation.We conducted extensive research on double mutations in Thermobida fusca to gain a deeper understanding of the enzyme's degradation capabilities. 203he activity of Cut190, a cutinase variant from S. viridis, was found to be inuenced by the presence of Ca 2+ in the binding site.Prior research has discovered three calcium binding sites in Cut190, each exerting distinct effects on the active site of the enzyme.By making modications to these sites, the thermal stability of the enzyme was enhanced, and the degradation of PET was greatly increased. 242The main function of PETase is to transform PET into intermediate compounds such as mono-(2hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET).These compounds are subsequently broken down by MHETase into ethylene glycol (EG) and terephthalic acid (TPA).These products are subsequently involved in the tricarboxylic acid (TCA) cycle.PETase functions best in a pH range of 7-9 and maintains its stability in a pH range of 6-10. 243An optimal pH of 9.0 and a temperature of 30 °C were determined to be the most effective conditions for the variant of PETase.Efforts to enhance the stability of Ideonella sakaiensis PETase involved targeted genetic modications to enhance its resistance to heat and prolong its effectiveness (Fig. 7). 244n a previous study, it was discovered that a specic mutation, R280A, demonstrated exceptional efficacy.This mutation showed enhanced activity and improved hydrolysis efficiency when BHET was used as a substrate.Additional examination of PETase resulted in the discovery of two mutations that enhance stability through the formation of hydrogen bonds: the substitution of serine at 247 position 121 with glutamic or aspartic acid, and the substitution of aspartate at position 186 with histidine.The double mutation (serine 121 and aspartate 186) led to the development of a PETase R280A variant that exhibited a remarkable enhancement in degradation capacity, resulting in a 13.9-fold increase in efficiency. 182Recent research has also investigated the alteration of the protein structure to enhance plastic degradation.By integrating a poly-3-hydroxybutyrate (PBM) binding domain into the enzyme, its capacity to break down PET was greatly enhanced.With professional experimentation, it was discovered that incorporating a CBM domain into the mutant greatly enhanced the speed at which PET degradation occurred.In fact, the rate increased by 2.28 times when compared to the original variant.These advancements highlight the potential of engineered enzymes in effectively tackling the issue of PET microplastic pollution. 219,225proaches to address microplastic and nanoplastic pollution

Exploring the environmental dynamics of micro-and nanoplastics
With the rise in plastic production worldwide, it is concerning to see how plastics are breaking down into microplastics due to various environmental factors such as ultraviolet light, temperature uctuations, soil biological activity, and human involvement.Microplastics can undergo a process where they break down into even smaller particles called nanoplastics, which worsens the issue of plastic pollution.These microplastics can take the form of bers, pellets, spheres, or akes.
The presence of micro-and nanoplastics is widespread in different environments, including landlls, wastewater systems, industrial and agricultural wastes, and polymer coatings. 248,249An in-depth understanding of the sources, properties, and distribution of these microplastic particles is crucial in the context of enzyme-based microplastic degradation strategies. 250This knowledge is vital for developing efficient measures to minimize their environmental impact.The extensive dispersion of these pollutants and their challenging degradation and recycling underscore the pressing need to tackle this worldwide environmental issue.An allencompassing approach is required to take on the extensive dispersion of micro-and nanoplastics and minimize their effects on ecosystems.

Biological effects of micro-and nanoplastics
Recent studies have uncovered the profound effects of microplastics and nanoplastics on the biological functions and health of a wide range of organisms.These studies have highlighted the negative impacts on ecosystem health and physiological processes of living organisms.They have emphasized the detrimental impact of large quantities of microplastics on the regular functioning of aquatic organisms, [251][252][253] particularly sh.These minuscule particles, consumed through food and inhalation, result in harm to the tissues of sh.Giacomo Limonta's research on zebrash revealed noteworthy alterations in the expression of immune response genes following exposure to low concentrations of microplastics.In Mehdi Banaei's research on Cyprinus carpio, signicant ndings were observed regarding gene expression and enzyme activity associated with oxidative stress and detoxication.][256] Microplastics present a signicant danger to various forms of marine life, extending beyond just sh.8][259][260] The risks are further intensied by the combined effects of manufacturing chemicals and organic contaminants that are attached to microplastics.Microplastics also support the development of various microbial communities and create biolms made up of algae, bacteria, and fungi.This occurrence has the potential to amplify the transmission of microbial pathogens and antimicrobial resistance. 261,262he potential health effects of microplastics on humans are currently receiving signicant attention.It has been estimated that a considerable quantity of microplastics is consumed by humans annually, potentially resulting in various health issues including intestinal blockages, inammatory reactions, and alterations in the gut microbiome.This growing research highlights the importance of further investigating the environmental and health effects of microplastics and nanoplastics. 263,264In particular, there is an urgent demand for the advancement and improvement of enzyme-based techniques to effectively break down these prevalent pollutants.

Using microbial capabilities for plastic degradation
The search for microorganisms and enzymes capable of degrading plastics is gaining importance in the battle against plastic pollution.This requires the application of molecular cloning and culture techniques to improve the enzymatic and metabolic abilities of these microorganisms, thereby enhancing their potential for plastic degradation. 107The use of molecular biology tools, specically polymerase-based rapid cloning, has been key in the discovery of polymer-degrading enzymes and the mapping of the genes responsible for these functions.These tools enable the manipulation of gene expression through genetic engineering to improve enzyme production, resulting in more efficient degradation in both the natural environment and composting facilities. 265xtensive microbial libraries have been developed to identify and validate microbes that excel at degrading plastics.Studies of the 16S rRNA gene in these libraries have yielded valuable insights into the microbial ecosystems linked to plastics.They have shed light on the interactions between these communities and factors such as substrate type, geographic location, and seasonal variations.The degradation of plastics by microbes is inuenced by various factors, including the chemical composition of the polymer, environmental conditions, and the inherent characteristics of the microbes themselves.The chemical composition of the polymer is important in determining its biodegradability, with environmental conditions playing a supporting role in promoting degradation.Microbial enzymes are crucial in this process since they selectively target substrates for biodegradation.It is necessary to understand the metabolic pathways of microorganisms that efficiently degrade polymers to develop targeted strategies to combat microplastic pollution.This involves studying bacteria and fungi that play a key role in this process.This occurrence has the potential to amplify the transmission of microbial pathogens and antimicrobial resistance. 266,267he potential health effects of microplastics on humans are currently being closely examined.It is estimated that people consume substantial quantities of microplastics annually, resulting in potential health issues such as intestinal blockages, inammatory reactions, and alterations in the gut microbiome.This emerging research highlights the importance of ongoing investigation into the environmental and health effects of microplastics and nanoplastics.There is an urgent demand for the advancement and improvement of enzyme-based techniques to effectively break down these prevalent pollutants.

Conclusions
It is imperative that we address the pressing issue of plastic pollution caused by the widespread presence of microplastics and nanoplastics with immediate and efficient measures.This review has emphasized the signicant role of enzymatic and microbial strategies in tackling these global environmental and health challenges.The use of enzymes such as PETase and MHETase, along with microbial degradation pathways, presents exciting possibilities for breaking down tough polymers such as PE, PET, and PS into more environmentally friendly substances.Despite the notable progress made in understanding and enhancing the capabilities of specic enzymes and microbes, there are still challenges that need to be addressed.Factors to consider are the efficiency of the degradation process, the scalability of these solutions, and the varying properties of plastic polymers.The signicance of microplastics on the environment and health, specically on marine life and human health, underscores the importance of implementing efficient degradation and recycling technologies.A combination of disciplines is needed to address plastic pollution and nd effective solutions.Further studies and developments are necessary to enhance the effectiveness and real-world implementation of enzymatic and microbial degradation methods.Enzymatic and microbial strategies show great potential in addressing plastic pollution.

Scheme 1
Scheme 1 Schematic diagram of microbial and enzymatic degradation and upcycling of microplastic.Microplastics in the environment undergo enzymatic degradation by extracellular enzymes and are then utilized as a carbon source by microorganisms, ultimately leading to complete.Figures generated with BioRender(https://biorender.com/).
Scheme 1 Schematic diagram of microbial and enzymatic degradation and upcycling of microplastic.Microplastics in the environment undergo enzymatic degradation by extracellular enzymes and are then utilized as a carbon source by microorganisms, ultimately leading to complete.Figures generated with BioRender(https://biorender.com/).

Fig. 1
Fig. 1 Enzymatic degradation of various types of plastics.Microorganisms inhabiting various surfaces of microplastics release various types of extracellular enzymes for biodegradation and fragmentation.Figures generated with BioRender(https://biorender.com/).
Fig. 1 Enzymatic degradation of various types of plastics.Microorganisms inhabiting various surfaces of microplastics release various types of extracellular enzymes for biodegradation and fragmentation.Figures generated with BioRender(https://biorender.com/).

Fig. 2
Fig. 2 Schematic diagram of the increase in microplastic decomposition efficiency using microbial engineering.Microorganisms discovered in various waste treatment facilities are ultimately improved for efficient microplastic degradation through various processes such as protein engineering, metagenomic library techniques, and bioreactor design.Figures generated with BioRender(https:// biorender.com/).
Fig. 2 Schematic diagram of the increase in microplastic decomposition efficiency using microbial engineering.Microorganisms discovered in various waste treatment facilities are ultimately improved for efficient microplastic degradation through various processes such as protein engineering, metagenomic library techniques, and bioreactor design.Figures generated with BioRender(https:// biorender.com/).

Fig. 3
Fig. 3 Microplastic decomposition using microbial engineering.(a) Construction of LCC-expressing plasmid pHK-LCC and extracellular expression of active LCC in C. thermocellum.Reproduced with permission from ref. 36.Copyright (2020) John Wiley & Sons, Inc.(b) Schematic of the designer T-E consortium composed of two strains Pp-T and Pp-E.Pp-T specializes in TPA degradation, which was developed by deleting the ped operon and constitutively expressing the genes tpaAa, tpaAb, tpaB, tpaC, and tpaK.Reproduced with permission from ref. 132.Copyright (2023) Springer Nature.(c) Cocultivation of Y. lipolytica Po1fP and P. stutzeri TPA3P.(A) OD, glucose consumption, and PHB content.(B) BHET hydrolysis curve.Reproduced with permission from ref. 133.Copyright (2021) Elsevier.(d) Biofilm formation and viability.Morphotypes of the cells in the mature biofilm on the PE sheet.Fluorescent microscopic images of biofilms, which show cell viability after the 28 day incubation.Reproduced with permission from ref. 110.Copyright (2014) American Chemical Society.

Fig. 4
Fig. 4 Schematic diagram of protein engineering strategies employed in the modification of enzymes involved in plastic degradation.The incorporation of protein engineering in the enhancement of plasticdegrading enzymes can induce alterations in enzyme activity and characteristics via diverse mechanisms.This encompasses the refinement of thermal stability, augmentation of enzyme activity through electrostatic interactions between the enzyme and substrate, and the potentiation of enzyme activity through the integration of accessory.

Fig. 5
Fig. 5 Strategies for enhancing thermal stability and catalytic activity of plastic degrading enzymes using protein engineering.(a) The IsPETase mutant, characterized by increased thermal stability and a higher T m value compared to the wild-type IsPETase, achieves this improvement through the stabilization of the b6-b7 linked loop.Reproduced with permission from ref. 181.Copyright (2019) American Chemical Society.(b) Enhanced catalytic activity of a PETase-EKn variant with a more open substrate binding pocket resulting from Cterminal fusion of PETase with a zwitterionic polypeptide consisting of glutamic acid (E) and lysine (K) residues.Reproduced with permission from ref. 185.Copyright (2021) American Chemical Society.(c) PETase with a narrow active site due to the double mutation S238F/W159H shows a higher loss of crystallinity in PET and improved aromatic interaction with the substrate compared to wild-type PETase.Reproduced with permission from ref. 186.Copyright (2018) PNAS.(d) Synergistic depolymerization efficacy of MHETase-PETase, a chimeric enzyme linking the C terminus of MHETase to the N terminus of PETase, on PET films.Reproduced with permission from ref. 186.Copyright (2020) PNAS.
binding module (CBM) of T. fusca cellulase Cel6A (CBM(Cel6A)) and Cellulomonas mi cellulase CenA (CBM(CenA)) to the C terminus of T. fusca cutinase Fusing the CBM module to the cutinase C terminus Increase of the amount of released fatty acids up to 3-fold compared with the wcompost genome PET Fusing the chitin binding domain (ChBD) of Chitinolyticbacter meiyuanensis SYBC-H1 to the C terminus of the LCC ICCG Fusing the ChBD to the C terminus of the LCCICCG variant -end fusion of bifunctional lipase-cutinase (Lip-Cut) Fusing the lipase and the cutinase Increase of the weight loss of PCL lm by 13.3 times, 11.8 times, and 5.7 times higher than that caused by Lip, Cut, and Lip/Cut blends, respectively 221 MHETase:PETase Ideonella sakaiensis PET Covalently links the C terminus of MHETase to the N terminus of PETase Fusing the MHETase and the PETase Increase of hydrolysis product release rate by 3-fold compared with the single enzyme (MHETase) 222

Fig. 6
Fig. 6 Proposed mechanism for PET degradation in Ideonella sakaiensis.The extracellular PETase enzyme catalyzes the hydrolysis of PET, yielding MHET as a byproduct.MHETase, predicted to be a lipoprotein, further hydrolyzes MHET into TPA and EG.Both TPA and EG can serve as energy sources for Ideonella sakaiensis and other microbes.Figures generated with BioRender(https://biorender.com/).
Fig. 6 Proposed mechanism for PET degradation in Ideonella sakaiensis.The extracellular PETase enzyme catalyzes the hydrolysis of PET, yielding MHET as a byproduct.MHETase, predicted to be a lipoprotein, further hydrolyzes MHET into TPA and EG.Both TPA and EG can serve as energy sources for Ideonella sakaiensis and other microbes.Figures generated with BioRender(https://biorender.com/).

Fig. 7
Fig. 7 Increased PET decomposition efficiency by PETase deformation.(a) The SEM images (up panel) and water contact angle analysis (down panel) of the PET film in a single-enzyme degradation system, two-enzyme degradation system with DBsEst, and a two-enzyme degradation system with DChryBHETase after 48 h at 60 °C.Reproduced with permission from ref. 245.Copyright (2023) Springer Nature.(b) HPLC profiles of PETase powder incubation experiments: 2 weeks, 3 weeks and 4 weeks after incubation.Green and red lines indicate CC-124 wild type and CC-124_PETase #11 lysates.Reproduced with permission from ref. 246.Copyright (2020) Springer Nature.(c) PETase activity of the variants, PET degradation activity of IsPETaseWT and variants, the enzyme activity is the sum of MHET and TPA.Reproduced with permission from ref. 181.Copyright (2019) American Chemical Society.(d) Enzyme activity for 10 days and heatinactivation experiment of IsPETaseWT and IsPETaseS121E/D186H/ R280A.Reproduced with permission from ref. 181.Copyright (2019) American Chemical Society.

Table 2
Examples of different protein engineering strategies of plastic-degrading enzymes to improve thermostability and biocatalytic performance